A.H. Zwinderman

The role of a common variant of the cholesteryl ester transfer protein gene in the progression of coronary atherosclerosis. The Regression Growth Evaluation Statin Study Group

BACKGROUND The high-density lipoprotein (HDL) cholesterol concentration is inversely related to the risk of coronary artery disease. The cholesteryl ester transfer protein (CETP) has a central role in the metabolism of this lipoprotein and may therefore alter the susceptibility to atherosclerosis. METHODS The DNA of 807 men with angiographically documented coronary atherosclerosis was analyzed for the presence of a polymorphism in the gene coding for CETP. The presence of this DNA variation was referred to as B1, and its absence as B2. All patients participated in a cholesterol-lowering trial designed to induce the regression of coronary atherosclerosis and were randomly assigned to treatment with either pravastatin or placebo for two years. RESULTS The B1 variant of the CETP gene was associated with both higher plasma CETP concentrations (mean [+/-SD], 2.29+/-0.62 microg per milliliter for the B1B1 genotype vs. 1.76+/-0.51 microg per milliliter for the B2B2 genotype) and lower HDL cholesterol concentrations (34+/-8 vs. 39+/-10 mg per deciliter). In addition, we observed a significant dose-dependent association between this marker and the progression of coronary atherosclerosis in the placebo group (decrease in mean luminal diameter: 0.14+/-0.21 mm for the B1B1 genotype, 0.10+/-0.20 mm for the B1B2 genotype, and 0.05+/-0.22 mm for the B2B2 genotype). This association was abolished by pravastatin. Pravastatin therapy slowed the progression of coronary atherosclerosis in B1B1 carriers but not in B2B2 carriers (representing 16 percent of the patients taking pravastatin). CONCLUSIONS There is a significant relation between variation at the CETP gene locus and the progression of coronary atherosclerosis that is independent of plasma HDL cholesterol levels and the activities of lipolytic plasma enzymes. This common DNA variant appears to predict whether men with coronary artery disease will benefit from treatment with pravastatin to delay the progression of coronary atherosclerosis.

Biological and chemical monitoring of occupational exposure to ethylene oxide.

Studies were carried out on two populations occupationally exposed to ethylene oxide (EtO) using different physical and biological parameters. Blood samples were collected from 9 hospital workers (EI) and 15 factory workers (EII) engaged in sterilization of medical equipment with EtO and from matched controls (CI and CII). Average exposure levels during 4 months (the lifespan of erythrocytes) prior to blood sampling were estimated from levels of N-(2-hydroxyethyl)valine adducts in hemoglobin. They were significantly enhanced in EI and EII and corresponded to a 40-h time-weighted average of 0.025 ppm in EI and 5 ppm in EII. Exposures were usually received in bursts with EtO concentrations in air ranging from 22 to 72 ppm in EI and 14 to 400 ppm in EII. All samples were analyzed for HPRT mutants (MFs), chromosomal aberrations (CAs), micronuclei (MN) and SCEs. MFs were significantly enhanced by 60% in EII but not in EI. These results are the first demonstration of mutation induction in man by ethylene oxide. CAs were significantly enhanced in EI and EII by 130% and 260% respectively. MN were not enhanced in EI but significantly in EII(217%). The mean frequency of SCEs was significantly elevated by 20% in EI and by almost 100% in EII. SCE was the only parameter that allowed distinction between daily and occasionally exposed workers in EII. An interesting finding in exposed workers was the large increase of the percentage of cells with high frequencies of SCE (3-4 times in EI and 17-fold in EII). The relative sensitivity of endpoints for detection of EtO exposure in the present investigation was in the following order: HOEtVal adducts greater than SCEs greater than chromosomal aberrations greater than micronuclei greater than HPRT mutants.

Measurement of frequencies of HPRT mutants, chromosomal aberrations, micronuclei, sister-chromatid exchanges and cells with high frequencies of SCEs in styrene/dichloromethane-exposed workers

Frequencies of HPRT mutants (MFs), chromosomal aberrations with or without gaps (CA+; CA-), aberrant cells (AC), micronuclei (MN), sister-chromatid exchanges (SCEs) and cells with high frequencies of SCEs (HFCs) were measured in lymphocytes collected from 46 workers occupationally exposed to styrene and dichloromethane (DCM = methylene chloride). These parameters were also determined in 23 controls. Time-weighted average (TWA) values for styrene and DCM exposure during an 8-h working day were respectively 70 mg/m3 (range: 0-598) and 108 mg/m3 (range: 0-742). These values correspond to TWA values of 17 ppm styrene and 31 ppm DCM. In exposed workers, all cytogenetic parameters were significantly enhanced (P < 0.0001; one-sided), but, due to the lack of appropriate control data, no definite conclusions could be drawn concerning the mutagenicity of styrene/DCM exposure. Duration of exposure was not correlated with genetic effects analyzed. The TWA value for styrene was not correlated with the extent of genetic damage detected, but the TWA value for DCM was positively correlated with the frequencies of chromosome aberrations (with gaps) and aberrant cells. These observations make it difficult to decide whether styrene or DCM, or both chemicals, induced the cytogenetic effects observed in exposed workers. Using the present styrene/DCM data, earlier ethylene oxide data and unpublished epichlorohydrin data, the relative sensitivity of the genetic endpoints to detect genotoxic exposure was: HFC > CA- > CA+ > SCE > MN > HPRT.

Chemotherapy-induced chromosomal damage in peripheral blood lymphocytes of cancer patients supplemented with antioxidants or placebo.

A total of 27 patients with various types of cancer were treated with cisplatin-based combination chemotherapy. Out of these, 13 patients were randomized to receive supplementation treatment with a beverage containing the antioxidants vitamins C and E, plus selenium, during chemotherapy. The antioxidant mixture was administered to investigate whether it could reduce the potential genotoxic and nephrotoxic effect of the applied chemotherapy. A placebo group of 14 cancer patients received a beverage without selenium or antioxidants. Micronuclei (MN) in cytochalasin B-blocked binucleate (BN) peripheral blood lymphocytes (PBLs) and hypoxanthine phosphoribosyl transferase (HPRT) mutants in PBLs were studied before, during and after chemotherapy as a measure for chemotherapy-induced genotoxic effects. Before chemotherapy, patients mean frequencies of MN and HPRT mutants did not differ from those in a group of 10 healthy subjects. The mean frequency of MN in patients increased significantly after one cycle of chemotherapy (P=0.002). This frequency was still elevated at 2 months after the completion of chemotherapy (not significantly). There was no significant difference in micronuclei frequency (MNF) between the antioxidant and placebo group of patients. Chemotherapy-induced frequencies of MN after three cycles of chemotherapy correlated significantly with the cumulative dose of cisplatin (r=0.58, P=0.012) and the cisplatin-mediated loss of renal function (r=0.53, P=0.03). No consistent change in HPRT mutant frequency following chemotherapy was observed in the placebo and antioxidant group of patients. In conclusion, cisplatin-combination chemotherapy resulted in a cisplatin dose-related increase of the frequency of chromosomal damage. Supplementation with antioxidants did not prevent or reduce this effect.

Frequencies of HPRT mutants and micronuclei in lymphocytes of cancer patients under chemotherapy: a prospective study

Fifteen cancer patients, including 10 testicular carcinoma patients, were treated with several types of combination chemotherapy. Blood samples were collected before, during and after chemotherapy. Subsequently, lymphocytes were analyzed for frequencies of HPRT mutants (MF) and micronuclei (MNF). Significantly elevated MFs were detected in eight patients. Mean expression time (+/- SD) for mutations was 98 +/- 54 days (range: 42-172 days). In some patients, enhanced MFs persisted for a period of 430-490 days after cessation of chemotherapy. In five patients MNFs were increased 2-6-fold and the enhancement was fairly persistent. Ifosfamide and cyclophosphamide appeared to be the most mutagenic and clastogenic constituents of the chemotherapy, while evidence for adverse effects of adriamycin, 4-epi-adriamycin and bleomycin was equivocal. Results indicate that the clinical use of mutagenic drugs must be weighed against the risks of persistent genetic damage and secondary malignancies in cured patients and their potential offspring. Further studies are necessary to determine the true risks and incidence of such abnormalities following chemotherapy for curable forms of cancer.

Estimation of multilocus haplotype effects using weighted penalised log-likelihood: Analysis of five sequence variations at the cholesteryl ester transfer protein gene locus

Direct analyses of haplotype effects can be used to identify those specific combinations of alleles that are associated with a specific phenotype. We introduce a method for direct haplotype analysis that solves two problems that arise when haplotypes are analysed in populations of unrelated subjects. Instead of assigning a single, most likely, haplotype pair to multiple heterozygous subjects, all haplotype pairs compatible with their genotype were determined and the posterior probabilities of these pairs were calculated using Bayes’ theorem and estimated haplotype frequencies. For the individual patients, all possible haplotype pairs were included in the statistical analysis using the posterior probabilities as weights, which were re‐estimated in an iterative process together with the haplotype effects. The second problem of unstable haplotype effect estimates, due to the numerous haplotypes and the low frequency at which some occur, was solved by assuming that haplotypes sharing the same alleles show a similar effect and that the extent of this similarity relates to the number of alleles shared. These assumptions were incorporated in a weighted log‐likelihood model by introducing a penalty, where differences in effects of similar haplotypes were penalised. Using CETP gene haplotypes, consisting of five closely linked polymorphisms, and baseline CETP and HDL‐C concentrations from the REGRESS population, we demonstrated that the model resulted in more stable effects than estimates based on unambiguous patients only.

TNFa Microsatellite Polymorphism Modulates the Risk of IDDM in Caucasians With the High-Risk Genotype HLA DQA1*0501-DQB1*0201/DQA1*0301-DQB1*0302

IDDM is a genetically controlled autoimmune disease. In particular, the loci of the HLA region on chromosome 6p are associated with the genetic risk for developing IDDM. DQAl*0501-DQBl*0201/DQAl*0301DQB1*O3O2 is the most prevalent susceptibility genotype in Caucasians, carrying a relative risk between 20 and 50 (1). However, the frequency of this high-risk genotype in the population is 10-20 times higher than the prevalence of IDDM associated with this genotype, suggesting that additional protective genes (HLA or non-HLA) and/or environmental factors can influence susceptibility to the disease (1). Recently, it has been shown that DRBl*0403 protects against IDDM in Caucasians with the high-risk heterozygous DR3-DQ2/DR4-DQ8 genotype (2), but it accounts for only -10% of the protection. Thus, DRB alleles alone cannot explain either protection or susceptibility for IDDM. To define additional markers for genetic susceptibility to or protection against IDDM in the HLA-DQ high-risk genotype group, 120 diabetic patients and 83 nondiabetic control subjects, all sharing the DQAl*0501-DQBl*0201/DQAl*0301DQBl*0302 genotype, were studied with eight microsatellite loci spanning the entire major histocompatibility complex (MHC) region and a region 2 cM telomeric of MHC. All subjects were unrelated European Caucasians and were recruited by the Belgian Diabetes Registry (2,3). HLA-typing and amplification of microsatellite markers were performed as described previously (3-5). Primer sequences for the markers HLA-F, D6S265, TNFa, D6S273, D6S1014, DQCar, TAP1, and D6S291 are available from the Genome Data Bank and were used as described previously (5). Microsatellite

Comparison of induction of hprt mutations by 1,3-butadiene and/or its metabolites 1,2-epoxybutene and 1,2,3,4-diepoxybutane in lymphocytes from spleen of adult male mice and rats in vivo

Induction of hprt mutations by 1,3-butadiene (BD) and its metabolites 1,2-epoxybutene (EB) and 1,2,3,4-diepoxybutane (DEB) was studied in lymphocytes from spleens of 6- to 14-week-old mice and 10- to 11-week-old rats. For unknown reasons, results from experiments with mice that received inhalation exposure to BD were quite variable. In the first experiment, mice were exposed for 5 days to 200, 500 or 1300 ppm and this resulted in a statistically significant, dose-dependent, induction of mutations. When the experiment was repeated and an extra expression time for mutations was included, it was not possible to detect induction of mutations. In a third experiment, a 6-day exposure to 500 ppm was mutagenic when mice with zero mutants were not excluded from the statistical analysis of the data. The monofunctional metabolite EB appeared to be mutagenic in mice (3 x 33 and 3 x 100 mg/kg), but not in rats (3 x 33 and 100 mg/kg or 30 days drinking water with 0.1, 0.3, or 1.0 mM EB). Contrary to expectations, there was no induction of mutations in mice and rats exposed to the bifunctional metabolite DEB (mice, 3 x 7, 21, 3 x 14, or 42 mg/kg; rats, 20 or 40 mg/kg or 30 days drinking water with 0.3 or 1 mM DEB), although in our earlier studies with mice and rats, DEB treatment significantly enhanced frequencies of micronuclei in splenocytes and in early spermatids of mice and rats. Some of these results differ from findings reported by other investigators. It is now becoming evident that these differences are, to a large extent, due to differences in age of the animals at the time of treatment. For example, the mutagenic potency of BD, EB and DEB was stronger in preweanling mice or 4-week-old mice than in 8- to 12-week-old adult mice.

Measurement of HPRT mutations in splenic lymphocytes and haemoglobin adducts in erythrocytes of Lewis rats exposed to ethylene oxide

Young adult male Lewis rats were exposed to ethylene oxide (EO) via single intraperitoneal (i.p.) injections (10-80 mg kg-1) or drinking water (4 weeks at concentrations of 2, 5, and 10 mM) or inhalation (50, 100 or 200 ppm for 4 weeks, 5 days week-1, 6 h day-1) to measure induction of HPRT mutations in lymphocytes from spleen by means of a cloning assay. N-ethyl-N-nitrosourea (ENU) and N-(2-hydroxyethyl)-N-nitrosourea (HOENU) were used as positive controls. Levels of N-(2-hydroxyethyl)valine (HOEtVal) adducts in haemoglobin (expressed in nmol g-1 globin) were measured to determine blood doses of EO (mmol kg-1 h, mM h). Blood doses were used as a common denominator for comparison of mutagenic effects of EO administered via the three routes. The mean HPRT mutant frequency (MF) of the historical control was 4.3 x 10(-6). Maximal mean MFs for ENU (100 mg kg-1) and HOENU (75 mg kg-1) were 243 x 10(-6) and 93 x 10(-6), respectively. In two independent experiments, EO injections led to a statistically significant dose-dependent induction of mutations, with a maximal increase in MF by 2.3-fold over the background. Administration of EO via drinking water gave statistically significant increases of MFs in two independent experiments. Effects were, at most, 2.5-fold above the concurrent control. Finally, inhalation exposure also caused a statistically significant maximal increase in MF by 1.4-fold over the background. Plotting of mutagenicity data (i.e., selected data pertaining to expression times where maximal mutagenic effects were found) for the three exposure routes against blood dose as common denominator indicated that, at equal blood doses, acute i.p. exposure led to higher observed MFs than drinking water treatment, which was more mutagenic than exposure via inhalation. In the injection experiments, there was evidence for a saturation of detoxification processes at the highest doses. This was not seen after subchronic administration of EO. The resulting HPRT mutagenicity data suggest that EO is a relatively weak mutagen in T-lymphocytes of rats following exposure(s) by i.p. injection, in drinking water or by inhalation.

D6STNFa Microsatellite Locus Correlates with CTLp Frequency in Unrelated Bone Marrow Donor-Recipient Pairs

The use of unrelated donors for bone marrow transplantation is associated with an increased morbidity and mortality when compared with HLA identical siblings, primarily due to an increased rate of graft-versus-host-disease. HLA matching for donors and recipients is the most important factor influencing the outcome of BMT. However, unrelated donor selection generally relies on matching only for HLA antigens without considering potential incompatibility for other MHC loci. Cellular assays have been developed to predict incompatibility that cannot be detected by current typing methods. The CTLp frequencies correlate with the degree of incompatibility of patient/donor and the clinical grade of GVHD. Since the CTLp assay is expensive and time consuming, an alternative is wanted. We studied the means of matching for microsatellites in determining MHC identity and possible correlation with CTLp frequencies. Therefore, 26 recipient/donor pairs were analysed for eleven microsatellite loci within and around the MHC region. Our study provides evidence that the D6STNFa locus correlates with CTLp frequency. The D6STNFa locus provides an additional marker that may help to improve the matching of unrelated donors and bone marrow recipients.